There are a number of toxins, including botulinum neurotoxin, ricin, shiga toxin and Staphylococcus enterotoxin B (SEB), that have either been used as bioterrorism agents or are considered a bioterrorism threat because of their extreme toxicity or ease of availibility. Furthermore, several of these toxins (botulinum neurotoxin, ricin) work at low concentrations, requiring highly sensitive assays for detection. Additionally, it is important to distuiguish between active and inactive toxin because of the possibility that genetically engineered toxins, consisting of the enzymatic portion of the toxin and a binding domain of another protein, can be used as the bioweapon agent. Consequently, our long-term goal is to develop diagnostic assays for toxin detection and differentiation that address these dual requirements of high sensitivity and determination of toxin activity. Our objective in this proposal is to develop a platform for toxin detection based upon a combination of two analytical approaches: 1) molecular recognition based upon ELISA microarrays and 2) enzymatic activity assays for toxin activity. We propose that this combined platform will provide a highly sensitive and specfic assay for the detection of these toxins. The specific aims are: 1). Develop an ELISA microarray for the sensitive and quantitative detection of botulinum neurotoxins (serotypes A, B, C, D, E, and F), ricin toxin, shiga toxin and Staphylococcal enterotoxin B. 2). Functional activity assays specific for botulinum neurotoxins, ricin, and shiga toxin will be developed. 3). A single platform, comprised of the ELISA microarray and the activity assay will be developed, allowing for the sensitive and specific detection and the determination of functional activity of, botulinum neurotoxins, ricin, Shiga toxin and Staphylococcal enterotoxin B in clinical samples. The combined assay platform will be used to analyze clinical samples, including sera, nasal swabs and stool samples, and for the determination of the sensitivity and specificity of the assays. RELEVANCE (See instructions): In the event of a bioweapon attack, it will be critical to rapidly determine which bioweapon agent is responsible for the attack in order to give the appropiate medical care. Our objective in this proposal is to develop a sensitive and specific assay for the detection of the following toxins: botulinum neurotoxin, ricin toxin, shiga toxin and Staphylococcus enterotoxin B.